0001] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S.

Provisional Patent Application No. 62/444,735, filed on January 10, 2017, to U.S. Provisional Patent Application No. 62/444,756, filed on January 10, 2017, to U.S. Provisional Patent Application No. 62/467,945, filed on March 7, 2017, to U.S. Provisional Patent Application No. 62/488,251, filed on April 21, 2017, to U.S. Provisional Patent Application No. 62/488,407, filed on April 21, 2017, to U.S. Provisional Patent Application No. 62/510,019, filed on May 23, 2017, and to U.S. Provisional Patent Application No. 62/510, 024, filed on May 23, 2017, the disclosures of which are incorporated herein by reference in their entireties.

FIELD

[0002] The instant application relates to (among other things), multi-arm polymer conjugates of Toll-like receptor ("TLR") agonists, and in particular, Toll-like receptor agonists of TLR 7 and/or TLR 8, as well as to compositions comprising the multi-arm polymer TLR agonist conjugates, and methods of making and using the conjugates. The instant application also relates to the field of cancer immunotherapy and involves, for example, the treatment of an individual having cancer by administering to the individual a toll-like receptor (TLR) agonist, e.g., a multi-arm polymer conjugate of a toll-like receptor agonist, in combination with a long-acting IL-2RP-biased agonist, and related compositions and methods, to be described in greater detail herein.

BACKGROUND

[0003] Toll-like receptors ("TLRs") are expressed on several cell types belonging to the innate and adaptive immune system. At least 13 different TLRs have been identified to date in mammals (Zhao, G., et al., Journal for ImmunoTherapy of Cancer 2014, 2: 12). TLR1, -2, -4, -5, -6, and -10 are expressed on the cell surface, while TLR 3, -7, -8, and -9 are situated on endosomal membranes within the cell (Kaczanowska, S., et al., J. Leukoc Biol. 2013 Jun; 93(6): 847-863). TLRs are sensors detecting pathogen and malignant cell-derived molecules called

pathogen-associated molecular patterns (PAMPs) which, upon binding to TLRs, trigger the ( F)-KB and type I interferon pathways resulting in the production of pro-inflammatory cytokines in dendritic cells (DCs) and other antigen presenting cells such as macrophages. TLRs are crucial for stimulation of DC maturation, antigen uptake and presentation, and the differentiation of CD4+ cells and control of regulatory T (Treg) cells.

[0004] TLR agonists have been investigated for their antitumor properties, however, in general, most TLR agonists have unde erformed as cancer therapeutics. It has been postulated that such underperformance might be explained by a mechanism in which induction of immune suppressive factors dampens TLR agonist-induced inflammation. (Lu, H. Frontiers in

Immunology, March 2014, 5, 83). For example, TLR agonists have immune stimulatory effects through the induction of co-stimulatory molecules such as CD80, CD86, and CD40 on dendritic cells and inflammatory cytokines such as T F-α and IL-12 that polarize the immune response. However, TLR agonists also have immune inhibitory effects, e.g., by inducing several immune suppressive factors including IL-10, regulatory T cells (Tregs), and PD-1, all of which can suppress anti-tumor immunity (Lu, H., 2014, ibid).

[0005] TLRs-7, -8, and -9 are similar in their recognition of nucleic acid motifs and expression within endosomal compartments (Zhao, G., 2014, ibid). Several ligands, both synthetic and natural nucleosides, have beers characterized as TLR7 and/or TLRS ligands.

Recognition of these nucleoside ligands by TLR7 or TLRS receptors activates intracellular pathways that culminate in the induction of proinflammatory cytokines, chemokines, and type I interferons (TFNs), and in the upregulation of co-stimulatory molecules. TLRs are type I membrane proteins, characterized by an ectodomain composed of leucine-rich repeats, responsible for recognition of pathogen-associated molecular patterns, and a cytoplasmic domain, called the Toll/'mterleukm-l receptor (TiR) domain, which is required for downstream signaling. TLR7 and TLRS are closely related, sharing their intracellular endosomal location, as well as their ligands. Recognition of a ligand by TLR7 or TLRS is followed by recruitment of the TLR domain-containing adaptor molecule myeloid differentiation primary response gene 88 (MyD88). The association of TLR7/8 and MyD88 stimulates the recruitment of members of the interleukiii-1 receptor-associated kinase family, resulting in the downstream activation of mitogen -activated protein kinases (MAPKs) and the ϊκΒ kinase (IKK) complex. Toll-like

receptor agonists of TLR 7 and TLR 8 activate macrophages and can, in some instances, change the tumor environment from a tumor-promoting to a tumor-suppressive (inflammatory) environment.

[0006] In light of their potential ability to activate several cell types such as DCs, monocytes, macrophages, fibroblasts, and human keratinocytes, induce apoptosis, generate enhanced immunogenicity and sensitization to ki iling mediated by cytotoxic T-cell lymphocytes and chemotherapeutics, TLR ligands are considered to be a class of immune-response modifiers having the potential to generate an effective antitumor immune response. Furthermore, T.LR8 ligands have been shown to reverse the suppressive function of CD8÷ Treg cells (Kiniwa Y., et al ., Clin Cancer Res 2007, 13 : 6947-58). Moreover, the application of TLR8 ligands resulted in a reduction of tumor infiltrating Foxp3+ Treg cells changing the tumor environment from tumor promoting to tumor suppressive (Anz D. et al.. Cancer Res, 2015; 75: 4483-93). On the other hand, TLR activation has, in certain instances, been shown to be advantageous for the proliferation, invasiveness, and/or survival of tumor cells (see, e.g., Bohnhorst J., et al.,

Leukemia 2006; 20: 1 138-1144; and Jego G., et al., Leukemia 2006;20: 1130-1 137). Certain TLR 7/8 agonists have also been shown to induce immunosuppression and autoimmune disease (Chi FL, et al. Frontiers in Pharmacology. 2017; 8: 304).

[0007] Although there have been substantial efforts in developing new and improved

TLR agonists that overcome one or more of the above-noted drawbacks, there remains a need to identify and provide new and more effective TLR agonists and related treatment regimens that overcome the shortcomings of prior art compounds and existing treatment methodologies whilst also providing a favorable immune response without triggering significant undesirable side effects such as inflammation. The present disclosure seeks to address this and other needs. The TLR7/8 agonists described herein can be used as stand-alone iramunotherapeutics (i .e., as a mono-immunotherapeutic), or, in another aspect, can be used in combination with a long acting IL-2RP-biased agonist.

SUMMARY

[0008] In a first aspect, the disclosure is directed to a multi-arm polymer conjugate of a

Toll-like receptor ("TLR") agonist. More particularly, the conjugate comprises a TLR 7/8

agonist compound covalently attached, via a linkage-containing spacer moiety, to a multi-arm, water-soluble, non-peptidic polymer. Among other things, the conjugates provided herein allow local administration of the conjugate, e.g., to a tumor site, wherein the conjugate is effective to preferentially initiate anti-tumor immunity locally during residence at the tumor site. The architecture of the multi-armed conjugate, along with the particular TLR 7/8 agonist, attachment chemistry, and mode of administration are effective to result in a conjugate that remains for an extended period of time within a tumor, and is effective to increase tumor antigen presentation and T-cell stimulation (i.e., to result in enhanced CD8 T cell priming), that is, to elicit an innate immune response, while accompanied by minimal toxic side effects due to localized activity.

[0009] In some embodiments of the multi-arm conjugate, the TLR 7/8 agonist compound is a small molecule.

[0010] In yet one or more further embodiments of the multi-armed polymer conjugate, the multi-armed water-soluble polymer comprises from 3 to about 50 polymer arms, or from 3 to about 10 polymer arms, or from 3 to 6 polymer arms. In one or more particular embodiments, the multi-armed polymer conjugate comprises 3, 4, or 5 polymer arms. In one or more further embodiments, the multi-armed polymer conjugate comprises 4 polymer arms.

[0011] In some embodiments, the conjugate comprises a TLR 7/8 agonist covalently attached at the terminus of one or more of the arms of the multi-arm, water-soluble non-peptidic polymer. In one or more embodiments, the TLR 7/8 agonist is covalently attached at the terminus of each of the arms of the multi-arm, water-soluble non-peptidic polymer. In yet one or more embodiments, each of the polymer arms of the multi-armed polymer conjugate is the same.

[0012] In some particular embodiments, a multi-arm polymer conjugate has a structure in accordance with Formula I:

Formula I

wherein R, taken together with each Q, is a residue of a polyol, polythiol, or polyamine bearing from 3 to about 50 hydroxyl, thiol, or amino groups; each Q is a linker selected from oxygen, sulfur and - H (e.g., corresponding to an oxygen, sulfur or nitrogen atom from the polyol, polythiol, or polyamine, respectively); each POLY is independently a water-soluble, non-peptidic polymer; each Xr is independently a linkage-containing spacer moiety; q is a positive integer from 3 to about 50; and each TLR 7/8 AG is a Toll-like receptor 7/8 agonist; or is a pharmaceutically acceptable salt form thereof.

[0013] In yet one or more further embodiments, the TLR 7/8 agonist is (N-[4-(4-amino-2-ethyl-lH-imidazo[4,5c]quinolin-l-yl)butyl] methane sulfonamide or [8-(3-(pyrrolidin-l-ylmethyl)benzyl)-4-amino-2-butoxy-7,8-dihydropteridin-6(5H)-one].

[0014] In some other embodiments, the TLR 7/8 agonist is an imidazoquinoline compound. In one or more preferred embodiments, the TLR 7/8 agonist is resiquimod or imiquimod, or an analog, derivative, or isomer thereof.

[0015] In yet some further embodiments in reference to Formula I, R, taken together with

Q, is a residue of a polyol.

[0016] In some embodiments pertaining to Formula I, each Xr is independently a stable linkage-containing spacer moiety. In yet some alternative embodiments, each Xr is independently a releasable linkage-containing spacer moiety.

[0017] In some additional embodiments related to Formula I, q is a positive integer selected from 3 to 10, or is a positive integer selected from 3 to 6, or is a positive integer selected from 3, 4, and 5, or is 4.

[0018] In yet some further embodiments, the linkage-containing spacer moiety comprises a thioether, carbamate, ester, carbonate, or urea functional group.

[0019] In yet some additional embodiments, the linkage-containing spacer moiety comprises an enzyme-cleavable peptidic linkage.

[0020] In one or more particular embodiments pertaining to Formula I, Xr is in accordance with Formula II:

Formula II

where a is zero or one (meaning that when a is zero, X1 is absent, and when a is one, X1 is present); b is zero or one (meaning that when b is zero, Lr is absent, and when b is one, Lr is

present); X1, when present, is a spacer; Lr, when present, is a linkage; and X2 is a functional group directly covalently attached to the TLR 7/8 agonist.

[0021] In some embodiments related to Formula II, "a" is zero. In yet some other embodiments, "b" is zero. In some additional embodiments, both "a" and "b" are zero. In yet some further embodiments, "a" is one. In yet some further embodiments, "b" is one. In yet some additional embodiments, both "a" and "b" are one.

[0022] In some further embodiments related to Formula II, X1 is -CH2C(0)-.

[0023] In yet some additional embodiments related to Formula II, X2 is selected from the group consisting of -C(0)- H-, - H-C(0)- H-, - H-C(O)-, and -ML

[0024] In yet some additional embodiments related to Formula II, Lr is selected from the group consisting of -(CRxRy)z-, and - H(CRxRy)z-, where each Rx and Ry is independently selected from hydrogen, lower alkyl, halo, and halo- substituted lower alkyl, and z is an integer from 1 to 6. For example, in some additional particular embodiments of the foregoing, Lr is selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, - CH2CH2CH2CH2CH2CH2- -CH2CHF-, -CHCH3-, -CHCH(CH3)2-, -CHCH2CH(CH3)2-, -C(CH3)2-, - HCH2-, - HCH2CH2-, - HCH2CH2CH2-, - HCH2CH2CH2CH2-, - HCH2CH2CH2CH2CH2-, - HCH2CH2CH2CH2CH2CH2- -NHCH2CHF-, - HCHCFL-, - HCHCH(CH3)2-, - HCHCH2CH(CH3)2-, and - HC(CH3)2-.

[0025] In one or more embodiments of the multi-arm polymer portion of the conjugate, the water-soluble, non-peptidic polymer is a poly(alkylene oxide). In some particular embodiments, the poly(alkylene oxide) is a poly(ethylene oxide).

[0026] In some embodiments, the water-soluble, non-peptidic polymer comprised within each of "q" polymer arms contains from about 1 to about 30 monomelic subunits. In yet some other embodiments, the overall water-soluble, non-peptidic polymer, i.e., including each of its polymer arms, has a molecular weight of from about 2,000 Daltons to about 150,000 Daltons. In some certain other embodiments, the overall water-soluble, non-peptidic polymer has a molecular weight of from about 5,000 Daltons to about 40,000 Daltons. In yet additional embodiments, the overall water-soluble, non-peptidic polymer has a molecular weight of from about 5,000 Daltons to about 25,000 Daltons.

[0027] In one or more particular embodiments, the multi-arm polymer conjugate of a TLR

7/8 agonist has a formula in accordance with Formula III:

(Formula III)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein L is -(CH2)m- -(CH2)m- H-C(0)-(CH2)m-,-CHF-(CH2)m- H-C(0)-(CH2)m-, -CH(CH3)- H-C(0)-(CH2)m-, -(CH2)m-CH(CH(CH3)2)- H-C(0)-(CH2)m-, -(CH2)m-CH(CH2CH(CH3)2)- H-C(0)-(CH2)m-, -C(CH3)2- H-C(0)-(CH2)m-, a single bond, or - H-(CH2)m-, each m is independently an integer from 1 to 5, inclusive; each n is independently an integer from 40 to 350, inclusive; R1 is hydrogen or -CFh-O-CH2-CH3; and R2 is hydrogen or hydroxyl. In yet some further embodiments related to Formula III, L is selected from -CH2-, -CH2-CH2- H-C(0)-CH2- -CH2-CH2-CH2-, -CHF-CH2- H-C(0)-CH2- -CH2-NH-C(0)-CH2- -CH(CH3)- H-C(0)-CH2- -CH2-CH(CH(CH3)2)- H-C(0)-CH2- -CH2-CH(CH2CH(CH3)2)- H-C(0)-CH2-, -C(CH3)2- H-C(0)-CH2-, a single bond, and -NH-CH2-CH2-.

[0028] In yet some further embodiments of Formula III, each n is independently an integer from 100 to 250, inclusive. In yet some other embodiments, R1 is hydrogen and R2 is hydrogen. In yet some additional embodiments, R1 is -CH2-O-CH2-CH3 and R2 is hydroxyl.

[0029] In one or more particular embodiments, the multi-armed polymer conjugate is selected from Compounds 1-10 and 12-16 as follows:

compound 1 ^ wherein each

PEG5k is linear polyethylene glycol having a formula ~(CH2CH20)yCH2CH2~, where y is about 113;

Compound 2 Compound 3

Compound 4 Compound 5

Compound 6 Compound 7

Compound 8 Compound 9

Compound 10 Compound 12

Compound 13 Compound 14

Compound 15 Compound 16

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each n is independently an integer from 40 to 350.

[0030] In one or more particular embodiments, the multi-armed polymer conjugate is

Compound 6.

[0031] In yet one or more additional embodiments, provided is a composition comprising a multi-arm polymer conjugate as described herein and a pharmaceutically acceptable excipient.

[0032] In yet another aspect (e.g., a second aspect), the disclosure provides a method of treatment comprising administering a multi-arm polymer conjugate as provided herein to a subject in need thereof.

[0033] In yet another, i.e., third, aspect, disclosed is a multi-armed polymer conjugate of a

TLR 7/8 agonist as provided herein for use in the treatment of cancer.

[0034] In a related, i.e., fourth, aspect, disclosed is a multi-armed polymer conjugate of a

TLR 7/8 agonist as provided herein for use in the preparation of a medicament useful in the treatment of cancer.

[0035] In yet another, i.e., fifth, aspect, disclosed is a method of preparing a multi-armed polymer conjugate of a TLR 7/8 agonist by covalently attaching either via a stable or releasable linkage-containing spacer moiety, a TLR 7/8 agonist to a multi-armed water-soluble polymer under conditions suitable to effect said covalent attaching.

[0036] In yet a further, i.e., sixth, aspect, provided herein is a method comprising administering to a subject having cancer, a TLR agonist such as, for example, a multi-armed polymer conjugate of a TLR 7/8 agonist as disclosed herein and an IL-2RP-activating amount of a long acting IL-2RP-biased agonist, both to be described in greater detail herein. In certain embodiments, the combination is effective to promote activation of the immune system (for example through promotion of CD8 T cells, CD1 lc+ and CD8+ dendritic cells, and neutrophils), while also overcoming immune suppression (for example though suppression of T regulatory cells, macrophages, and monocytes).

[0037] By way of clarity, with regard to the sequence of administering, the TLR 7/8 agonist and the long acting IL-2RP-biased agonist may be administered concurrently or sequentially and in any order, and via the same and/or different routes of administration, each in an immunomodulating amount. Moreover, treatment may comprise a single cycle of therapy, or may comprise multiple (i.e., two or more) cycles of therapy.

[0038] In one or more embodiments, the TLR agonist is administered locally and the long acting IL-2RP-biased agonist is administered parenterally. In one or more related embodiments, the TLR agonist, e.g., a multi-armed polymer conjugate of a TLR 7/8 agonist, is administered directly to the site of a tumor.

[0039] In one or more embodiments related to the sixth aspect, the TLR agonist, e.g., a multi-armed polymer conjugate of a TLR 7/8 agonist, is administered to the subject separately from the long acting IL-2RP-biased agonist.

[0040] In yet one or more further embodiments, the TLR agonist, e.g., a multi-armed polymer conjugate of a TLR 7/8 agonist, is administered to the subject prior to administering the long acting IL-2RP-biased agonist. For example, in one or more embodiments, the TLR agonist and the long acting IL-2RP-biased agonist are both administered on day 1 of treatment. In one or more alternative embodiments, the TLR agonist is administered on day 1 of treatment and the long acting IL-2RP-biased agonist is administered on any one of days 1 to 4 of treatment. For example, the long acting IL-2RP-biased agonist is administered on any one of days 1, 2, 3, or 4 of treatment.

[0041] In a preferred embodiment, the subject is a human subject.

[0042] In one or more additional embodiments, the cancer is a solid cancer. For example, the cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric cancer, lymphoma, malignant melanoma, liver cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancers, kidney cancer, cancer of the bile duct, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease and adrenocortical cancer.

[0043] In some embodiments, the long-acting IL-2RP-biased agonist comprises aldesleukin releasably covalently attached to polyethylene glycol. In yet some additional embodiments, the long acting IL-2RP-biased agonist comprises aldesleukin releasably covalently attached to from 4, 5 and 6 polyethylene glycol polymers. In yet some further embodiments, the long acting IL-2RP-biased agonist comprises aldesleukin releasably covalently attached to an average of about 6 polyethylene glycol polymers. In one or more additional embodiments, the polyethylene glycol polymers that are releasably covalently attached to aldesleukin are branched.

[0044] In yet some further embodiments related to any one or more of the foregoing aspects or embodiments, the TLR agonist is a TLR 7 or a TLR 8 agonist. In one or more embodiments, the TLR agonist is a TLR 7 agonist. In yet one or more alternative embodiments, the TLR agonist is a TLR 8 agonist. In some embodiments, the TLR agonist is a long-acting TLR agonist such as a long acting TLR 7 or a long-acting TLR 8 agonist (e.g., a multi-armed polymer modified TLR 7 or TLR 8 agonist.

[0045] In yet some additional embodiments, the long-acting TLR agonist is a multi-armed water-soluble polymer conjugate of a TLR agonist such as a TLR 7/8 agonist. In yet one or more further embodiments, the multi-armed water-soluble polymer is stably covalently linked to the TLR agonist, e.g., the TLR 7/8 agonist. In one or more alternative embodiments, the multi-armed water-soluble polymer is releasably covalently linked to the TLR agonist, e.g., the TLR 7/8 agonist.

[0046] In yet one or more particular embodiments, the long-acting TLR agonist is a 4-arm-pentaerythritolyl-based polyethylene glycol conjugate having a TLR agonist molecule covalently linked, either stably or releasably, at the terminus of each of its four polymer arms.

[0047] In some preferred embodiments, the long acting IL-2RP-biased agonist comprises compounds encompassed by the following formula:

wherein IL-2 is an interleukin-2, "n" is an integer from about 3 to about 4000, or pharmaceutically acceptable salts thereof.

[0048] In some embodiments, the long acting IL-2RP-biased agonist having a formula as set forth in the preceding paragraph is comprised in a composition comprising no more than 10% (based on a molar amount) of compounds encompassed by the following formula:

wherein IL-2 is interleukin-2, n' is an integer selected from the group consisting of 1, 2, 3, 7 and >7, and pharmaceutically acceptable salts thereof.

[0049] In some further embodiments of the method of administering, the TLR 7/8 conjugate has the following structure:

wherein each n is independently an integer from 40 to 350. In one or more related embodiments, the value of n in each of the polymer arms is substantially the same. In some particular embodiments, the value of n in each of the four polymer arms is about 113.

[0050] In some embodiments of the sixth aspect, the administering is effective to produce an abscopal effect in the subject.

[0051] In some further embodiments related to the foregoing, the administering is effective to provide a percent survival rate, when evaluated in a suitable animal model, such as a mouse CT-26 colon tumor model, at a day after start of treatment that is after the day by which all subjects in the vehicle only group have reached 0% survival, e.g., between days 35 and 50, that is greater than that observed for administration of each of the single agents alone, i.e., the long-acting IL-2RP-biased agonist and the TLR agonist.

[0052] In yet another aspect, provided is a kit comprising an IL-2RP-activating amount of a long acting IL-2RP-biased agonist and an innate immunity activating amount of a TLR agonist, e.g., the multi-armed polymer conjugate of a TLR 7/8 agonist, accompanied by instructions for use in treating a subject having cancer.

[0053] In one or more embodiments of the kit, the long acting IL-2RP-biased agonist and the TLR agonist are comprised in a single composition for administration to the subject, where the single composition optionally comprises a pharmaceutically acceptable excipient.

[0054] In some alternative embodiments of the kit, the long acting IL-2RP-biased agonist and the TLR agonist are provided in separate containers, and the kit comprises instructions for administering the TLR agonist and the long-acting IL-2RP-biased agonist separately to the subject.

[0055] In some embodiments of the kit, both the long-acting IL-2RP-biased agonist and the TLR agonist are in solid form. In one or more related embodiments, each of the long acting

IL-2RP-biased agonist and the long acting TLR agonist are in a solid form suitable for reconstitution in an aqueous diluent.

[0056] In yet one or more further embodiments, each of the long acting IL-2RP-biased agonist and the TLR agonist is comprised within separate compositions each comprising a pharmaceutically acceptable excipient.

[0057] Additional embodiments of the present conjugates, compositions, methods, and the like will be apparent from the following description, examples, and claims. As can be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG. L is a plot showing primary tumor (TLR agonist inj ection site) volume versus days following initial dosing of mice treated with various interventions (vehicle, exemplary long acting IL-2RP-biased agonist, RSLAIL-2; an exemplary TLR agonist, 4-arm-PEG20k-CM-N-R848; and a combination of RSLAIL-2 and 4-arm-PEG20k-CM-N-R848) in a mouse colon carcinoma model (CT-26) as described in detail in Example 21.

[0059] FIG. 2 is a plot showing secondary tumor (remote, non-TLR agonist injection site) volume versus days following initial dosing of mice treated with various interventions in a mouse colon carcinoma model (CT-26) as described in detail in Example 21.

[0060] FIG. 3 is a plot of percent survival versus days following initial dosing for mice treated with various interventions (vehicle, exemplary long acting IL-2RP-biased agonist, RSLAIL-2; an exemplary TLR agonist, 4-arm-PEG20k-CM-N-R848; and a combination of RSLAIL-2 and 4-arm-PEG20k-CM-N-R848) in a mouse colon carcinoma model (CT-26) as described in detail in Example 21.

[0061] FIGs. 4A-4D are plots showing that that combination treatment with RSLAIL-2 and R848 leads to decreased or maintained tumor volume of the treated tumor for nine of the ten animals in the high dosage group, seven of the ten animals in the mid dosage group, and only one of the eight animals in the low dosage group by day 25 after treatment start, as described in detail in Example 23.

[0062] FIGs. 4E-4H are plots showing that combination treatment with RSLAIL-2 and

Compound 6 leads to decreased or maintained tumor volume of the treated tumor for nine of the ten animals in the high dosage group, nine of the ten animals in the mid dosage group, and (surprisingly) ten of the ten animals in the low dosage group by day 32 after treatment start, as described in detail in Example 23.

[0063] FIGs. 5A-5D are plots showing that combination treatment with RSLAIL-2 and

R848 leads to decreased or maintained tumor volume of the non-treated tumor for nine of the ten animals in the high dosage group, five of the ten animals in the mid dosage group, and only one of the eight animals in the low dosage group by day 25 after treatment start, as described in detail in Example 23.

[0064] FIGs. 5E-5H are plots showing that combination treatment with RSLAIL-2 and

Compound 6 leads to decreased or maintained tumor volume of the non-treated tumor for nine of the ten animals in the high dosage group, eight of the ten animals in the mid dosage group, and (surprisingly) ten of the ten animals in the low dosage group by day 32 after treatment start, as described in detail in Example 23.

[0065] FIG. 6A and 6B are graphs providing a comparison of tumor and plasma cytokine concentrations at 2 hours and at 6 hours, respectively, following treatment with Compound 6, as described in Example 18. The concentration of systemic cytokines was significantly less than that in the tumor for each of the cytokines measured (IL-6, KC/GRO, TNF- a, IL-Ιβ, IL-5, IFN-a, IFN-γ, IL-2 and IL-12p70).

DETAILED DESCRIPTION

Definitions

[0066] As used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0067] "Water soluble, non-peptidic polymer" indicates a polymer that is at least 35% (by weight) soluble, preferably greater than 70% (by weight), and more preferably greater than 95%

(by weight) soluble, in water at room temperature. Typically, an unfiltered aqueous preparation of a "water-soluble" polymer transmits at least 75%, more preferably at least 95%, of the amount of light transmitted by the same solution after filtering. It is most preferred, however, that the water-soluble polymer is at least 95% (by weight) soluble in water or completely soluble in water. With respect to being "non-peptidic," a polymer is non-peptidic when it has less than 35% (by weight) of amino acid residues.

[0068] The terms "monomer," "monomeric subunit" and "monomelic unit" are used interchangeably herein and refer to one of the basic structural units of a polymer. In the case of a homo-polymer, a single repeating structural unit forms the polymer. In the case of a co-polymer, two or more structural units are repeated— either in a pattern or randomly— to form the polymer. Preferred polymers are homo-polymers. The water-soluble, non-peptidic polymer comprises one or more monomers serially attached to form a chain of monomers. The polymer can be formed from a single monomer type (i.e., is homo-polymeric) or two or three monomer types (i.e., is co-polymeric).

[0069] A "polymer" as used herein is a molecule possessing from about 2 to about 2000 or more, e.g. from about 2 to about 4000, monomers. Specific polymers include those having a variety of geometries such as linear, branched, or forked, to be described in greater detail below.

[0070] "PEG" or "polyethylene glycol," as used herein, is meant to encompass any water-soluble poly(ethylene oxide). Unless otherwise indicated, a "PEG polymer" or any polyethylene glycol is one in which substantially all (preferably all) monomeric subunits are ethylene oxide subunits, though, the polymer may contain distinct end capping moieties or functional groups, e.g., for conjugation. PEG polymers can comprise one of the two following structures: "-(CEhCEhC n-" or "-(CH2CH20)n-iCH2CH2-," depending upon whether or not the terminal oxygen(s) has been displaced, e.g., during a synthetic transformation. As stated above, for the PEG polymers, the variable (n) (i.e., number of repeat units) ranges from about 2 to 2000, or from abaout 2 to 4000, and the terminal groups and architecture of the overall PEG can vary. When PEG further comprises a functional group for linking to, e.g., a small molecule drug, the functional group when covalently attached to a PEG polymer does not result in formation of an oxygen-oxygen bond (-0-0-, a peroxide linkage).

[0071] The terms "end-capped" or "terminally capped" are interchangeably used herein to refer to a terminal or endpoint of a polymer having an end-capping moiety. Typically, although not necessarily, the end-capping moiety comprises a hydroxy or C i-20 alkoxy or an alkaaryloxy group. Thus, examples of end-capping moieties include alkoxy (e.g., methoxy and, ethoxy), benzyloxy, as well as aryl, heteroaryl, cyclo, heterocyclo, and the like. In addition, saturated, unsaturated, substituted and unsubstituted forms of each of the foregoing are envisioned. Moreover, the end-capping group can also be a silane. The end-capping group can also advantageously comprise a detectable label. When the polymer has an end-capping group comprising a detectable label, the amount or location of the polymer and/or the moiety (e.g., active agent) of interest to which the polymer is coupled, can be determined by using a suitable detector. Such labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric moieties (e.g., dyes), metal ions, radioactive moieties, and the like. Suitable detectors include photometers, films, spectrometers, and the like. In addition, the end-capping group may contain a targeting moiety.

[0072] The term "targeting moiety" refers to a molecular structure that helps the conjugates to localize to a targeting area, e.g., help enter a cell, or bind a receptor. Preferably, the targeting moiety comprises a vitamin, antibody, antigen, receptor, DNA, RNA, sialyl Lewis X antigen, hyaluronic acid, sugars, cell-specific lectins, steroid or steroid derivative, RGD peptide, ligand for a cell surface receptor, serum component, or combinatorial molecule directed against various intra-or extracellular receptors. The targeting moiety may also comprise a lipid or a phospholipid. Exemplary phospholipids include, without limitation, phosphatidylcholines, phospatidylserine, phospatidylinositol, phospatidylglycerol, and phospatidylethanolamine. These lipids may be in the form of micelles or liposomes and the like. The targeting moiety may further comprise a detectable label or alternately a detectable label may serve as a targeting moiety. When a polymer conjugate has a targeting group comprising a detectable label, the amount and/or distribution/location of the polymer and/or the moiety (e.g., active agent) to which the polymer is conjugated can be determined by using a suitable detector. Such labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions, radioactive moieties, gold particles, quantum dots, and the like.

[0073] Molecular weight in the context of a water-soluble polymer, such as PEG, can be expressed as either a number average molecular weight or a weight average molecular weight.

Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques. Other methods for measuring molecular weight values can also be used, such as the use of end-group analysis or the measurement of colligative properties (e.g., freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number average molecular weight or the use of light scattering techniques, ultracentrifugation, or viscometry to determine weight average molecular weight. PEG polymers are typically polydisperse (i.e., number average molecular weight and weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.

[0074] "Branched," in reference to the geometry or overall structure of a polymer, refers to a polymer having two or more polymers "arms" extending from a branch point.

[0075] "Forked," in reference to the geometry or overall structure of a polymer, refers to a polymer having two or more functional groups (typically through one or more atoms) extending from a branch point.

[0076] A "branch point" refers to a bifurcation point comprising one or more atoms at which a polymer branches or forks from a linear structure into one or more additional arms.

[0077] The term "reactive" or "activated" refers to a functional group that reacts readily or at a practical rate under conventional conditions of organic synthesis. This is in contrast to those groups that either do not react or require strong catalysts or impractical reaction conditions in order to react (i.e., a "nonreactive" or "inert" group).

[0078] "Not readily reactive," with reference to a functional group present on a molecule in a reaction mixture, indicates that the group remains largely intact under conditions that are effective to produce a desired reaction in the reaction mixture.

[0079] A "protecting group" is a moiety that prevents or blocks reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. The protecting group may vary depending upon the type of chemically reactive group that is being protected as well as the reaction conditions to be employed and the presence of additional reactive or protecting groups in the molecule. Functional groups which may be protected include, by way of example, carboxylic acid groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups and the like. Representative protecting groups for carboxylic acids include esters (such as a p-methoxybenzyl ester), amides and hydrazides; for amino groups, carbamates (such as tert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters; for thiol groups, thioethers and thioesters; for carbonyl groups, acetals and ketals; and the like. Such protecting groups are well-known to those skilled in the art and are described, for example, in T.W. Greene and G.M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.

[0080] A functional group in "protected form" refers to a functional group bearing a protecting group. As used herein, the term "functional group" or any synonym thereof encompasses protected forms thereof.

[0081] A "releasable linkage" is a relatively labile bond that cleaves under physiological conditions, wherein the cleavage may occur by way o any of a number of different mechanisms. One type of exemplary releasable linkage is a hydrolysable bond, that is, one that cleaves upon reaction with water (i.e., is hydrolyzed), e.g., under physiological conditions, such as for example, hydrolysis of an amide bond such as an aromatic amide bond. The tendency of a bond to hydrolyze in water may depend not only on the general type of linkage connecting two atoms but also on the substituents attached to these atoms. Appropriate hydrolytically unstable or weak linkages may include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides, oligonucleotides, thioesters, and carbonates. Releasable linkages also include enzymatically releasable linkages, where an "enzymatically releasable linkage" means a linkage that is subject to cleavage by one or more enzymes. Additional types of release mechanisms include but are not limited to 1,6-benzyl elimination, β-elimination, and the like. While certain bonds may be considered to be stable or releasable, such characterization should be considered within the overall structure of a molecule or structural entity. In certain instances, a polymer conjugate containing a releasable bond is referred to as a prodrug, wherein upon cleavage of the releasable bond in vivo (i.e., under physiological conditions), the parent drug is released (or is eventually released, depending upon the number of polymeric moieties releasably attached to an active agent). A covalent "releasable" linkage, for example, in the context of a water soluble polymer such as polyethylene glycol that is covalently attached to an active moiety such as interleukin-2 or a TLR agonist such as resiquimod (also known as R848), is one that cleaves under physiological conditions to thereby release or detach a water soluble polymer from the active moiety, or to detach an active moiety from a water-soluble polymer.

[0082] A "stable" linkage or bond refers to a chemical bond that is substantially stable in water (e.g., under physiological conditions), that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages generally include but are not limited to the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, amines, and the like. Generally, a stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.

[0083] A "TLR 7/8 agonist" (or "TLR agonist") is any compound which is an agonist to

Toll-like receptor 7 and/or Toll-like receptor 8.

[0084] "Substantially" or "essentially" means nearly totally or completely, for instance,

95% or greater, more preferably 97% or greater, still more preferably 98%> or greater, even more preferably 99% or greater, yet still more preferably 99.9% or greater, with 99.99%) or greater being most preferred of some given quantity.

[0085] "Alkyl" refers to a hydrocarbon chain, ranging from about 1 to 20 atoms in length.

Such hydrocarbon chains are preferably but not necessarily saturated and may be branched or straight chain. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl, isopropyl, 3-methylpentyl, and the like. As used herein, "alkyl" includes cycloalkyl when three or more carbon atoms are referenced. An "alkenyl" group is an alkyl group of 2 to 20 carbon atoms with at least one carbon-carbon double bond.

[0086] The terms "substituted alkyl" or "substituted Cq-r alkyl" where q and r are integers identifying the range of carbon atoms contained in the alkyl group, denotes the above alkyl groups that are substituted by one, two or three halo atoms (e.g., F, CI, Br, I), trifluoromethyl, hydroxy, Ci-7 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, butyl, t-butyl, and so forth), C1-7 alkoxy, C1-7 acyloxy, C3-7 heterocyclyl, amino, phenoxy, nitro, carboxy, acyl, cyano, or the like. The substituted alkyl groups may be substituted once, twice or three times with the same or with different substituents.

[0087] "Lower alkyl" refers to an alkyl group containing from 1 to 7 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl.

[0088] "Lower alkenyl" refers to a lower alkyl group of 2 to 6 carbon atoms having at least one carbon-carbon double bond.

[0089] "Non-interfering substituents" are those groups that, when present in a molecule, are typically non-reactive with other functional groups contained within the molecule.

[0090] " Alkoxy" refers to an -O-R group, wherein R is alkyl or substituted alkyl, preferably

C1-C20 alkyl (e.g., methoxy, ethoxy, propyloxy, etc.), preferably C1-C7.

[0091] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a component that may be included in the compositions described herein and causes no significant adverse toxicological effects to a patient.

[0092] The term "aryl" means an aromatic group having up to 14 carbon atoms. Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, naphthalenyl, and the like. "Substituted phenyl" and "substituted aryl" denote a phenyl group and aryl group, respectively, substituted with one, two, three, four or five (e.g., 1-2, 1-3 or 1-4 substituents) chosen from halo (F, CI, Br, I), hydroxy, cyano, nitro, alkyl (e.g., Ci-6 alkyl), alkoxy (e.g., Ci-6 alkoxy), benzyloxy, carboxy, aryl, and so forth.

[0093] An exemplary conjugate, active moiety, or other suitably applicable chemical moiety as described herein is meant to encompass, where applicable, analogues, isomers, polymorphs, solvates, and pharmaceutically acceptable salt forms thereof.

[0094] "Pharmacologically effective amount," "physiologically effective amount," and

"therapeutically effective amount" are used interchangeably herein to mean the amount of an active agent, typically a polymer conjugate, that is needed to provide a desired level of active agent and/or conjugate in the bloodstream or in the target tissue. The precise amount may depend upon numerous factors, e.g., the particular active agent, the components and physical characteristics of the composition, intended patient population, patient considerations, and may readily be determined by one skilled in the art, based upon the information provided herein and available in the relevant literature.

[0095] A basic reactant or an acidic reactant described herein includes neutral, charged, and any corresponding salt forms thereof.

[0096] The term "patient," or "subject" as used herein refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a compound or composition or combination as provided herein, such as a cancer, and includes both humans and animals. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and preferably are human.

[0097] "Optional" or "optionally" means that the subsequently described circumstance may but need not necessarily occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0098] A "small molecule" as used herein refers to an organic compound typically having a molecular weight of less than about 1000.

OVERVIEW

[0099] The multi-arm water-soluble polymer- TLR 7/8 agonist conjugates described herein incorporate a number of innovative advances in drug design and treatment rationale that integrate into a novel, potentially safer and highly efficacious anti-cancer therapy. They are capable of innate immune system activation, and, when comprised of releasable linkages to the TLR agonist compound, are effective to release and retain an active TLR 7/8 agonist in an injected tissue such as a cancerous tumor. The conjugates, when administered intratum orally, are effective to activate local tumor antigen presentation to cytotoxic T cells and overcome immune suppressive signals in the tumor environment. The multi-arm water-soluble polymer scaffold contributes to an injected conjugate being primarily retained at the injected tumor site, whereby TLR 7/8 agonist dependent immune activation is highest in the treatment site with reduced systemic activity. A key advantage of such drug design over prior systemic small molecule TLR agonists is localized drug activity at the treated tumor and decreased systemic exposure reducing potential toxicities.

[00100] Additionally, a combination treatment as provided herein, in which a multi-arm water-soluble polymer-TLR 7/8 agonist conjugate is administered in combination with a long acting IL-2RP-biased agonist, stems from an inherent synergy of the local TLR 7/8 agonist polymer conjugate-driven anti-tumor innate immune activation and a systemic intratumoral

expansion of cytotoxic T cells by the long acting IL-2RP-biased agonist. As described herein, studies in multiple syngeneic tumor models show that optimization of pharmacokinetic and pharmacodynamic properties of the dual therapeutic combination results in a highly efficacious combination therapy that successfully couples anti-tumor innate and adaptive immune activation analogously to a natural pathogen driven immune response. Both treatment components activate complementary arms of the immune system to engage the entire immune activation cascade required for systemic tumor clearance. The combination therapy described herein is designed to synergistically elicit a safer and more effective anti-tumor immune response than either agent administered singly. These and other features will become apparent and are described in detail in the sections which follow.

Multi-arm Polymer Conjugates of a TLR 7/8 Agonist

[00101] As described above, provided herein are multi-arm polymer conjugates of a Tolllike receptor ("TLR") agonist compound, i.e., a TLR 7/8 agonist. In some particular embodiments, the multi-arm polymer conjugate has a structure in accordance with Formula I:

Formula I

wherein R, taken together with each Q, is a residue of a polyol, polythiol, or polyamine bearing from 3 to about 50 hydroxyl, thiol, or amino groups, respectively; each Q is a independently a linker selected from oxygen, sulfur and - H (e.g., corresponding to an oxygen, sulfur or nitrogen atom from the polyol, polythiol, or polyamine, respectively); each POLY is independently a water-soluble, non-peptidic polymer; each Xr is independently a linkage-containing spacer moiety; q is a positive integer from 3 to about 50; and each TLR 7/8 AG is a Toll-like receptor 7/8 agonist, wherein the Formula I also encompasses pharmaceutically acceptable salts thereof. We will now consider each of the various components of the multi-arm polymer conjugate of Formula I.

[00102] Considering Formula I, in one or more embodiments, the residue of the polyol, polythiol or polyamine, "R," used in connection with the multi-arm polymer is an organic radical-containing moiety possessing from about 3 to about 150 carbon atoms (e.g., from about 3 to about 50 carbon atoms). In some preferred embodiments, R when taken together with Q, that is, (R-Q)q, that is the polyol, polyamine or polythiol core molecule, comprises from 3 to about 25 carbon atoms, or from 3 to about 10 carbon atoms, e.g., such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The residue may contain one more heteroatoms (e.g., O, S, or N) in addition to those defined by Q. By residue, in reference to a polyol (or polyamine or polythiol), is meant the parent molecule following removal of one or more of its terminal hydrogen atoms, to provide an organic radical suitable for attachment to POLY.

[00103] As previously indicated, the residue of the polyol, polythiol or polyamine, "R-Q"q that forms the basis of the branching for the multi-armed conjugates provided herein, originates from a corresponding polyol, polythiol or polyamine. In one or more embodiments, the corresponding polyol, polythiol, or a polyamine bears at least three hydroxyl, thiol, or amino groups, respectively, available for polymer attachment. A "polyol" is a molecule comprising three or more hydroxyl groups. A "polythiol" is a molecule that comprises three or more thiol groups. A "polyamine" is a molecule comprising three or more amino groups.

[00104] In one or more embodiments, the polyol, polyamine or polythiol typically contains

3 to about 25 hydroxyl groups, or amino groups, or thiol groups, respectively, such as from 3 to about 10 (i.e., 3, 4, 5, 6, 7, 8, 9, or 10) hydroxyl, amino groups or thiol groups, respectively, preferably from 3 to about 8 (i.e., 3, 4, 5, 6, 7, or 8) hydroxyl, amino groups or thiol groups, respectively. In one or more embodiments, the number of atoms between each hydroxyl, thiol, or amino group will vary, although lengths of from about 1 to about 20 (e.g., from 1 to about 5) intervening atoms, such as carbon atoms, between each hydroxyl, thiol or amino group, are exemplary. In referring to intervening core atoms and lengths, -CH2- is considered as having a length of one intervening atom, -CH2CH2- is considered as having a length of two atoms, and so forth.

[00105] Exemplary polyols and polyamines have (Radical)-(OH)q and (Radical)-( H2)q structures, respectively, where (Radical) corresponds to an organic-containing radical and q is a positive integer from 3 to about 50. Note that, as described above, in Formula I, the variable "Q," when taken together with R, typically represents a residue of the core organic radical as described herein. That is to say, when describing polyols, polythiols and polymer amines, particularly by name, these molecules are referenced in their form prior to incorporation into a multi-armed polymer-containing structure (i.e., are referred to as their parent molecules). That is to say, when describing preferred organic core molecules, particularly by name, the core molecules are described in their precursor form, rather than in their radical form after removal of, for example, one or more protons. So, if for example, the organic core radical is derived from pentaerythritol, the precursor polyol possesses the structure C(CH2OH)4, and the organic core radical, together with Q, corresponds to C(CH20-)4, where Q is O. So, for example, for a conjugate of Formula I wherein R taken together with Q is a residue of the polyol, pentaerythritol C(CH2OH)4, a residue R together with Q corresponds to "C(CH20-)4", such that each of "q" polymer arms in the multi-armed polymer conjugate will emanate from each of the oxygen atoms of the pentaerythritol core or residue.

[00106] Illustrative polyols include aliphatic polyols having from 1 to 10 carbon atoms and from 3 to 10 hydroxyl groups, including for example, trihydroxyalkanes, tetrahydroxyalkanes, polyhydroxy alkyl ethers, polyhydroxyalkyl polyethers, and the like. Cycloaliphatic polyols include straight chained or closed-ring sugars and sugar alcohols, such as mannitol, sorbitol, inositol, xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol, dulcitol, facose, ribose, arabinose, xylose, lyxose, rhamnose, galactose, glucose, fructose, sorbose, mannose, pyranose, altrose, talose, tagitose, pyranosides, sucrose, lactose, maltose, and the like. Additional examples of aliphatic polyols include derivatives of glucose, ribose, mannose, galactose, and related stereoisomers. Aromatic polyols may also be used, such as l, l, l-tris(4'-hydroxyphenyl) alkanes, such as l,l, l-tris(4-hydroxyphenyl)ethane, 2,6-bis(hydroxyalkyl)cresols, and the like. Other core polyols that may be used include polyhydroxycrown ethers, cyclodextrins, dextrins and other carbohydrates (e.g., monosaccharides, oligosaccharides, and polysaccharides, starches and amylase).

[00107] Exemplary polyols include glycerol, trimethylolpropane, pentaerythritol, dipentaerythritol, tripentaerythritol, ethoxylated forms of glycerol, trimethylolpropane, pentaerythritol, dipentaerythritol, tripentaerythritol. Also, preferred are reducing sugars such as sorbitol and glycerol oligomers, such as diglycerol, triglycerol, hexaglycerol and the like. A 21-arm polymer can be synthesized using hydroxypropyl-P-cyclodextrin, which has 21 available hydroxyl groups. Additionally, a polyglycerol having an average of 24 hydroxyl groups is also included as an exemplary polyol.

[00108] Exemplary polyamines include aliphatic polyamines such as diethylene triamine,

Ν,Ν',Ν''-trimethyldiethylene triamine, pentam ethyl diethylene triamine, tri ethylene tetramine, tetraethylene pentamine, pentaethylene hexamine, dipropylene triamine, tripropylene tetramine, bis-(3-aminopropyl)-amine, bis-(3-aminopropyl)-methylamine, and N,N-dimethyl-dipropylene-triamine. Naturally occurring polyamines that can be used include putrescine, spermidine, and spermine. Numerous suitable pentamines, tetramines, oligoamines, and pentamidine analogs suitable for use are described in Bacchi et al. (2002) Antimicrobial Agents and Chemotherapy, 46(1):55-61, which is incorporated by reference herein.

CLAIMED:

1. A conjugate comprising a TLR 7/8 agonist covalently attached, via a linkage- containing spacer moiety, to a multi-arm, water-soluble, non-peptidic polymer.

2. The conjugate of claim 1, wherein the TLR 7/8 agonist is a small molecule.

3. The conjugate of claim 1 or claim 2, wherein the multi-armed water-soluble polymer comprises from 3 to about 50 polymer arms.

4. The conjugate of any one of claims 1 -3, comprising the TLR 7/8 agonist covalently attached at the terminus of one or more of the arms of the multi-arm, water soluble non- peptidic polymer.

5. The conjugate of claim 4, comprising the TLR 7/8 agonist covalently attached at the terminus of each of the arms of the multi-arm, water soluble non-peptidic polymer.

6. The conjugate of claim 4, in accordance with Formula I:

Formula I

wherein:

R, taken together with each Q, is a residue of a polyol, polythiol, or polyamine bearing from 3 to about 50 hydroxyl, thiol, or amino groups;

each Q is a linker selected from oxygen, sulfur and - H;

each POLY is independently a water-soluble, non-peptidic polymer;

each Xr is independently a linkage-containing spacer moiety;

q is a positive integer from 3 to about 50; and

each TLR 7/8 AG is a Toll-like receptor 7/8 agonist; or

a pharmaceutically acceptable salt form thereof.

7. The conjugate of any one of claims 1-6, wherein the TLR 7/8 agonist is (N-[4-(4-amino-2-ethyl-lH-imidazo[4,5c]quinolin-l-yl)butyl] methane sulfonamide or [8-(3-(pyrrolidin-l-ylmethyl)benzyl)-4-amino-2-butoxy-7,8-dihydropteridin-6(5H)-one].

8. The conjugate of any one of claims 1-6, wherein the TLR 7/8 agonist is an

imidazoquinoline compound.

9. The conjugate of any one of claims 1-6, wherein the TLR 7/8 agonist is resiquimod or imiquimod.

10. The conjugate of any one of claims 1-9, wherein R, taken together with Q, is a residue of a polyol.

11. The conjugate of any of claims 6-10, wherein each Xr is independently a stable linkage-containing spacer moiety.

12. The conjugate of any of claims 6-10, wherein each Xr is independently a releasable linkage-containing spacer moiety.

13. The conjugate of any of claims 6-12, wherein q is a positive integer from 3 to 10.

14. The conjugate of any of claims6 - 12, wherein q is a positive integer from 3 to 6.

15. The conjugate of any of claims 6-12, wherein q is selected from 3, 4, and 5.

16. The conjugate of any of claims 6-12, wherein q is 4.

17. The conjugate of any one of the preceding claims, wherein the linkage-containing spacer moiety comprises a thioether, carbamate, ester, carbonate, or urea functional group.

18. The conjugate of any one of claims 1-16, wherein the linkage-containing spacer moiety comprises an enzyme-cleavable peptidic linkage.

18. The conjugate of any one of the preceding claims, wherein Xr is in accordance with Formula II:

-[XVtLrjb-X2- Formula II

where:

a is zero or one;

b is zero or one;

X1, when present, is a spacer;

Lr, when present, is a linkage; and

X2 is a functional group directly covalently attached to the TLR 7/8 agonist.

19. The conjugate of claim 18, wherein X1 is present.

20. The conjugate of claim 19, wherein X1 is -CH2C(0)-.

21. The conjugate of claim 18, wherein X1 is absent.

22. The conjugate of any one of claims 18-21, wherein X2 is selected from the group consisting of -C(0)- H-, - H-C(0)- H-, - H-C(O)-, and -ML

23. The conjugate of any one of claims 18-22, wherein Lr is present.

24. The conjugate of claim 23, wherein Lr is selected from the group consisting of -(CRxRy)z-, and - H(CRxRy)z-, where each Rx and Ry is independently selected from hydrogen, lower alkyl, halo, and halo-substituted lower alkyl, and z is an integer from 1 to 6.

25. The conjugate of claim 24, where Lr is selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2- -CH2CHF-, -CHCH3-, -CHCH(CH3)2-, -CHCH2CH(CH3)2-, -C(CH3)2-, - HCH2-, - HCH2CH2-, - HCH2CH2CH2-, - HCH2CH2CH2CH2-, - HCH2CH2CH2CH2CH2-, - HCH2CH2CH2CH2CH2CH2- - HCLhCHF-, - HCHCft-, - HCHCH(CH3)2-, - HCHCH2CH(CH3)2-, and -NHC(CH3)2-.

26. The conjugate of any one of the preceding claims, wherein the water-soluble, non-peptidic polymer is a poly(alkylene oxide).

27. The conjugate of claim 26, wherein the poly(alkylene oxide) is a poly(ethylene oxide).

28. The conjugate of any one of the preceding claims, wherein the water-soluble, non-peptidic polymer comprised within each of "q" polymer arms contains from about 1 to about 30 monomelic subunits.

29. The conjugate of any one of the preceding claims, wherein the water-soluble, non-peptidic polymer has a molecular weight of from about 2,000 Daltons to about 150,000 Daltons.

30. The conjugate of any one of the preceding claims, wherein the water-soluble, non-peptidic polymer has a molecular weight of from about 5,000 Daltons to about 40,000 Daltons.

31. The conjugate of any one of the preceding claims, wherein the water-soluble, non-peptidic polymer has a molecular weight of from about 5,000 Daltons to about 25,000 Daltons.

32. The conjugate of claim 1, having a formula in accordance with Formula III:

(Formula III)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

L is -(CH2)m- -(CH2)m- H-C(0)-(CH2)m- ,-CHF-(CH2)m- H-C(0)-(CH2)m--CH(CH3)- H-C(0)-(CH2)m- -(CH2)m-CH(CH(CH3)2)- H-C(0)-(CH2)m--(CH2)m-CH(CH2CH(CH3)2)- H-C(0)-(CH2)m- -C(CH3)2- H-C(0)-(CH2)m- a single bond, or - H-(CH2)m-

each m is independently an integer from 1 to 5, inclusive;

each n is independently an integer from 40 to 350, inclusive;

R1 is hydrogen or -CH2-0-CH2-CH3; and

R2 is hydrogen or hydroxyl.

33. The conjugate of claim 32, wherein L is -CH2- -CH2-CH2- H-C(0)-CH2--CH2-CH2-CH2- -CHF-CH2- H-C(0)-CH2- -CH2- H-C(0)-CH2--CH(CH3)- H-C(0)-CH2- -CH2-CH(CH(CH3)2)- H-C(0)-CH2--CH2-CH(CH2CH(CH3)2)- H-C(0)-CH2- -C(CH3)2- H-C(0)-CH2- a single bond, or

- H-CH2-CH2-.

34. The conjugate of claim 31 or claim 32, wherein each n is independently an integer from 100 to 250, inclusive.

35. The conjugate of any one of claims 32 to 34, wherein R1 is hydrogen and R2 hydrogen.

36. The conjugate of any one of claims 32-34, wherein R1 is -CH2-O-CH2-CH3 and R2 hydroxyl.

37. A conjugate selected from the group consisting of Compounds 1-10 and 12-16:

Compound 2 Compound 3

Compound 4 Compound 5

Compound 6 Compound 7

Compound 8 Compound 9

Compound 10 Compound 12

Compound 13 Compound 14

Compound 15 Compound 16

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each n is independently an integer from 40 to 350.

38. A composition comprising a conjugate of any one of the preceding claims and a pharmaceutically acceptable excipient.

39. A method of treatment comprising administering a conjugate or composition or composition of any one of of the preceding claim to a subject in need thereof.

40. The conjugate of any of claims 1-37 for use in the treatment of cancer.

41. The conjugate of any of claims 1-37 for use in the preparation of a medicament useful in the treatment of cancer.

42. A method of administration comprising administering to a subject having cancer (i) a conjugate comprising a TLR 7/8 receptor activating amount of a TLR 7/8 agonist covalently attached, via a linkage-containing spacer moiety, to a multi-arm, water-soluble, non-peptidic polymer according to any one of claims 1-37, and (ii) an IL-2RP-activating amount of a long acting IL-2RP-biased agonist.

43. The method of claim 42, wherein the conjugate and the long acting IL-2RP-biased agonist are administered concurrently or sequentially and in any order, and via the same and/or different routes of administration, each in an immunomodulating amount.

44. The method of claim 43, wherein the method comprises a single cycle of administering.

45. The method of claim 43, wherein the method comprises a two or more cycles of administering.

46. The method of any one of claims 42-45, wherein the conjugate is administered locally and the long acting IL-2RP-biased agonist is administered parenterally.

47. The method of claim 46, wherein the conjugate is administered directly to the site of a cancerous tumor (i.e., intratumorally).

48. The method of any one of claims 42-47, where the conjugate is administered to the subject separately from the long acting IL-2RP-biased agonist.

49. The method of any one of claim 42-49, wherein the conjugate is administered to the subject prior to administering the long acting IL-2RP-biased agonist.

50. The method of any one of claims 42-49, wherein the conjugate and the long acting IL-2RP-biased agonist are both administered on day 1 of treatment.

51. The method of any one of claims 42-49, wherein the conjugate is administered on day 1 of treatment and the long acting IL-2RP-biased agonist is administered on any one of days 1 to 4 of treatment.

52. The method of claim any one of claims 42-51, wherein the subject is a human subject.

53. The method of any one of claims 42-52, wherein the cancer is a solid cancer.

54. The method of claim 53, wherein the solid cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric cancer, lymphoma, malignant melanoma, liver cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancers, kidney cancer, cancer of the bile duct, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease and adrenocortical cancer.

55. The method of any one of claims 42-54, wherein the long-acting IL-2RP-biased agonist comprises aldesleukin releasably covalently attached to polyethylene glycol.

56. The method of claim 55, wherein the long acting IL-2RP-biased agonist comprises aldesleukin releasably covalently attached to from 4, 5 and 6 polyethylene glycol polymers.

57. The method of claim 56, wherein the long acting IL-2RP-biased agonist comprises aldesleukin releasably covalently attached to an average of about 6 polyethylene glycol polymers.

58. The method of claim 57, wherein the polyethylene glycol polymers that are releasably covalently attached to aldesleukin are branched.

59. The method of claim 58, wherein the long acting IL-2RP-biased agonist comprises compounds encompassed by the following formula:

wherein IL-2 is an interleukin-2, where "n" is an integer from about 3 to about 4000, or pharmaceutically acceptable salts thereof.

60. The method of claim 59, wherein the long acting IL-2RP-biased agonist is comprised in a composition comprising no more than 10% (based on a molar amount) of compounds encompassed by the following formula

wherein IL-2 is interleukin-2, n' is an integer selected from the group consisting of 1, 2, 3, 7 and >7, and pharmaceutically acceptable salts thereof.

61. The method of any one of claims 42-60, wherein the conjugate has the following structure:

wherein each n is independently an integer from 40 to 350.

62. The method of claim 61, wherein the value of n in each of the polymer arms is substantially the same.

63. The method of claim 61, wherein the value of n in each of the polymer arms is about

113.

64. The method of any one of claims 42-63, wherein the administering is effective to produce an abscopal effect in the subject.

65. The method of any one of claims 42-64, wherein the administering is effective to provide a percent survival rate, when evaluated in a suitable animal model, such as a mouse CT-26 colon tumor model, at a day after start of treatment that is after a day by which all subjects in the vehicle only group have reached 0% survival that is greater than that observed for administration of either of the long-acting IL-2RP-biased agonist or the conjugate alone, when administered at therapeutically equivalent doses to those used in the combination.

66. A combination for use in treating a subject having cancer, the combination comprising an IL-2RP-activating amount of a long acting IL-2RP-biased agonist and an innate immunity activating amount of a conjugate in accordance with any one of claims 1-37, accompanied by instructions for use in treating a subject having cancer.

67. The combination of claim 66, wherein the long acting IL-2RP-biased agonist comprises aldesleukin releasably covalently attached to polyethylene glycol.

68. The combination of claim 67, wherein the long acting IL-2RP-biased agonist comprises aldesleukin releasably covalently attached to from 4, 5 and 6 polyethylene glycol polymers.

69. The combination of claim 68, wherein the long acting IL-2RP-biased agonist comprises aldesleukin releasably covalently attached to an average of about 6 polyethylene glycol polymers.

70. The combination of any one of claims 67-69, wherein the polyethylene glycol polymers that are releasably covalently attached to aldesleukin are branched.

71. The combination of claim 66, wherein the long acting IL-2RP-biased agonist comprises compounds encompassed by the following formula:

wherein IL-2 is interleukin-2, where "n" is an integer from about 3 to about 4000,

or pharmaceutically acceptable salts thereof.

72. The combination of claim 71, wherein the long acting IL-2RP-biased agonist is comprised in a composition comprising no more than 10% (based on a molar amount) of compounds encompassed by the following formula

wherein IL-2 is interleukin-2, n' is an integer selected from the group consisting of 1, 2, 3, 7 and >7, or pharmaceutically acceptable salts thereof.

73. The combination of any one of claims 66-72, wherein the conjugate has the following structure:

wherein each n is independently an integer from 40 to 350.

74. The combination of claim 73, wherein the value of n in each of the polymer arms is substantially the same.

75. The combination of claim 73, wherein the value of n in each of the polymer arms is about 113.